This invention relates generally to mutants of Hepatitis B Virus (HBV), and more particularly, relates to new mutants of HBV, their significance in clinical applications, their use as reagents in detection of HBV infection and immunity and their use in vaccines.
HBV is known to cause a variety of disease states, from mild subclinical infection to chronic active and fulminant hepatitis. The hepatitis B genome is a circular, partially double stranded DNA of approximately 3200 base pairs which code for seven viral proteins. P. Tiollais et al., Nature 317:489-495 (1985). The polymerase gene completely overlaps the viral envelope genes PreS1, PreS2 and S, and partially overlaps the X and core genes. The envelope of the hepatitis B virion consists of three proteins and their glycosylated derivatives. These proteins, termed small (S), middle (M) and large (L) hepatitis B surface (HBs) proteins contain the S gene sequence. W. H. Gefiich et al., in Viral Hepatitis and Liver Disease, F. B. Hollinger et al., eds. Williams-Wilkens, Baltimore, Md., pages 121-134 (1991) The MHBs contains the PreS2 sequence (55 amino acids [a.a.]and the L protein contains the PreS1 sequence (108 or 119 a.a., depending on subtype) plus the PreS2 sequence. Only a very small portion of the total hepatitis B surface antigen exists as complete virions or Dane particles. Two other morphological forms, 22 nm spherical particals and filaments of 22 nm diameter and variable length, lack capsid or DNA and are produced in high excess over HBV virions.
The core gene encodes the nucleocapsid protein (183 or 185 a.a.), hepatitis B core Antigen (HBcAg). Immediately upstream of the core gene is the precore region which consists of 87 nucleotides encoding 29 a.a. in phase with the core gene. The first 19 a.a, of the precore region serve as a signal for membrane translocation and eventual secretion of the precore gene product, termed HBeAg. The function of HBe is enigmatic but may help the virus escape immune surveillance by inducing immune tolerance.
Because of the genomic compactness and the extensive functional overlaps, it is expected that significant constraints on DNA sequence divergence would occur in order to maintain a genome capable of efficient replication and transmission. The hepatitis B virus, however, shows greater mutability than previously appreciated. Similar to the Human Immunodeficiency Virus (HIV), HBV uses reverse transcriptase (RT) as an essential step in the replication cycle. RT has poor proofreading ability, leading to a high rate of nucleotide misincorporation. Calculations suggest that this error-prone replication leads to one point replacement, deletion or insertion per 1000 to 100,000 nucleotides copied. W. Carman et al., Lancet 341:349-353 (1993). Variability in the virus was first discovered through classical subtyping studies of HBsAg. A. M. Courouce et al., Bibliotheca Haematologica 42:1 (1976).
Mutations may not be located randomly on the genome. Recent reports have documented the emergence of other mutations in the pre core, core and envelope protein-genes, PreS and S, which presumably give these mutants a selective advantage over wild type (WT) in evading, the immune system.
Evidence suggests that vital clearance and liver cell injury in HBV infection are mediated by a cytotoxic T lymphocyte (CTL) response to one or more HBV-encoded antigens expressed at the hepatocyte surface. M. Peters et at., Hepatology 13:977-994 (1991). A strong T-cell response to HBcAg and HBeAg antigens, but not to envelope antigens, was found in acute hepatitis B. Persistence of vital replication correlated with a blunted T-cell response to HBcAg. Although T cell response to vital antigen may be abrogated in chronic HBV, CTL response may persist in chronic carriers.
Also, there is evidence of ongoing humoral response in both symptomatic and asymptomatic hepatitis B carriers. High levels of anti-HBc are observed in almost all HBV carriers. Twenty percent of random HBsAg positive specimens have detectable HBeAg and anti-HBe, and 10-20% have detectable but low level anti-HBs. A recent report using very sensitive detection methods indicates that virtually all HBV patients with liver disease and about 50% of chronic hepatitis B patients without liver disease have demonstrable humoral immune responses specific for HBeAg and anti-HBsAg and PreS Ag. Much of the anti-HBs response, however, may exist because of the different fine specificities of HBsAg and anti-HBs and probably is not neutralizing. These data support the theory that there is ongoing immune surveillance of precore, core and envelope gene products in chronic HBV carriers which could provide selective pressure for the emergence of HBV variants.
The HBV envelope regions encompassing PreS 1 and PreS2 and the region 120-160 a.a. of S are exposed on the surface of the vital particles, and thus would be expected to be targets of immune surveillance. W. H. Gerlich et al., supra. Some S mutants described to date have signicantly affected the antigenicity of the "a" epitope(s) which is common or group-specific determinant(s) of SHBs. W. Carman et al., Gastroenterology 102:711-719 (1992). The "a" determinants are complex, conformational and dependent upon disulfide bonding among highly conserved cysteine residues. The "a" immunoreactivity can be partially mimmicked by cyclic synthetic peptides. Although the "a" epitope(s) traditionally had been defined by reactivity to polyclonal antisera, the use of monoclonal antibody has shown that the "a" region consists of at least five non-overlapping epitopes. D. Peterson et al., J. Immunol. 132:920-927 (1984). Genetic variation in the "a" determinant leading to immune escape has been described in vacinees in Italy and Japan and in liver transplant patients on monoclonal anti-a antibody therapy. See, for example, W. F. Carman et al., Lancet 336:325-329 (1990); H. Okamoto et al., Pediatric Research 32:264-268 (1992); G. McMahon et al., Hepatology 15:757-766 (1992); H. Fujii et al., Biochem. Biophys. Res. Comm. 184:1152-1157 (1992); and T. J. Harrison et al., J. of Hepatology 13:5105-5107 (1991 ). The most common mutant described to date is a single nucleotide substitution leading to replacement of a glycine with an arginine (G-R 145). This mutation destroys some but not all "a" epitopes. Detection of anti-HBs with monoclonal antibody has not been problematic.
Other mutations in the "a" region lead to loss of subtypic or type specific determinants, y/d and w/r. Several recent papers have documented the emergence of gross deletions and point mutations in the PreS 1/PreS2 region suggesting that the production of these envelope gene products also are under immune selection in chronically infected individuals. HBV mutants which cannot replicate because of deletions in the env, C or P genes have been reported in plasma from HBV carders. All coexist with HBV forms which are replication competent. Okamoto et al. (Supra) demonstrated that mutant genomes with gross deletions in the PreS/S, C and P genes derived from plasma or asymptomatic carders may be complemented in transient expressions system with hepatoma cells. Complementation was measured as the ability to secrete viral particles with mutant genomes into the culture media. Interestingly, all mutants had an intact encapsidation signal. Complementation with predecessor WT viruses, other mutants and even with HBV DNA sequences integrated into host chromosomes was demonstrated in this in vitro system. Thus, the suggestion that HBV mutants acting as defective interfering particles may attenuate WT virus replication and thereby help maintain persistence of infection has been made.
The detection of mutants of Hepatitis B surface antigen therefore is important. Mutants may develop over time due to such factors as vaccine administration or infection. The identification and detection of mutant Hepatitis B Virus(es) may lead to vaccine development and detection systems to determine the presence of these mutants in test samples. A need therefore exists not only to identify these mutant strains, but also to provide detection systems capable of determining the presence of the mutant in a test sample. A further need also exists for the development of a vaccine to such mutant strain(s).